The multiple enzymatic activities of DNA polymerases must be carefully orchestrated and regulated to ensure accurate and efficient DNA synthesis during DNA replication and repair, while preventing the formation of mutagenic or unstable DNA intermediates. There is a fundamental gap in understanding functional coordination, which exists across the entire DNA polymerase family. The long-term goal is to understand in molecular detail how DNA polymerases function during DNA replication and repair. The objective of this application, which is the next step in the pursuit of this goal, is to determine ow the multiple enzymatic activities of E. coli DNA polymerase I (Pol I) are coordinated and regulated. Pol I is a readily manipulated model enzyme that exemplifies the essential features of multifunctional DNA polymerases from more complex organisms. The central hypothesis is that functional coordination is linked to the conformational dynamics of Pol I. In particular, it is proposed that the flexibly tethered 5' nuclease domain, through its ability to dynamically shift position, both orchestrates and enhances the different enzymatic activities of Pol I. This hypothesis has been formulated on the basis of preliminary data from the applicant's laboratory. The rationale for the proposed research is that once the conformational states of the 5' nuclease domain and its influence on the other domains are known, it will be possible to understand how Pol I coordinates it's different activities and why this coordination sometimes fails, leading to mutations, strand breaks or other deleterious outcomes. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: (1) Dissect the nucleotide incorporation cycle of Pol I and reveal the role of the 5' nuclease domain. (2) Dissect the 3'-5' exonuclease pathway of Pol I and reveal the role of the 5' nuclease domain. (3) Determine how the 5' nuclease activity is physically coordinated with the other enzymatic activities of Pol I. A range of novel single- molecule fluorescence spectroscopic methods will be developed in order to visualize conformational changes that mediate functional coordination in the Pol I enzyme, and the single-molecule data will be corroborated in parallel biochemical experiments. The approach is innovative, because it will employ a fundamentally new experimental approach, based on direct visualization of enzyme conformational changes within a single polymerase molecule as it switches between different modes of activity during a single encounter with a DNA substrate. The proposed research is significant, because it will establish an unprecedented level of understanding of how the different enzymatic activities of a multifunctional DNA polymerase work together during DNA replication and repair.